The liver is a vital regenerative organ that is composed of two cell types: hepatocytes and duct cells. Understanding how these cells are formed during regeneration and development are important in developing clinical therapies for the myriad of liver diseases and injuries. Currently, there is a significant gap in understanding the initial stages of liver development. The goal of this project is to gain a more thorough understanding of the murine liver bud, the morphologically distinct structure that harbors the liver progenitors. The liver bud emerges from the endoderm in a process that is typical of all endoderm-derived organs including the lung, pancreas and thyroid, using secreted signals from adjacent tissues. Aim 1 is to test the role of candidate signaling pathways in liver budding. To down-regulate the pathway of interest in the liver precursor population, two approaches utilizing whole embryo culture are used: electroporation of constructs designed to downregulate candidate pathways in localized endodermal domains and the addition of inhibitors directly to the culture media. The manipulated embryos are then cultured through the liver bud phase of development. If a candidate plays a role in budding then this process will be disrupted and its role inferred from the resultant phenotype. A novel Cre-expressing mouse line will be produced to test the role of validated candidates in vivo. Aim 2 is to test the hypothesis that the two liver precursor populations are distinct precursors. Fate mapping experiments have demonstrated that the liver is derived from two discreet populations of cells that contribute differently to the liver bud. Preliminary data demonstrate that the two populations are molecularly and morphologically distinct from one another and from the remainder of the endoderm. To assess the molecular differences between the two liver precursor populations and a third non-liver precursor, transcriptional profiles derived from pools of individually confirmed cells will be produced and compared. Aim 3 is designed to test the potential of individual liver bud cells, also termed hepatoblasts. Indirect evidence suggests that hepatoblasts are multipotent, giving rise to both hepatocytes and ductal cells. We propose a genetic marking strategy, utilizing an endoderm-specific CreER line and the R26R reporter, to produce single recombination events in the liver bud that turns on the reporter in that cell and all of its descendants. This retrospective lineage analysis will demonstrate how the liver grows and if the two liver cell-types are derived from a common liver bud precursor. Combined these three Aims will provide novel information on normal liver development that will greatly aid in understanding diseases of the liver and contribute to studies designed to induce hepatocytes and hepatic organogenesis in vitro. PUBLIC HEALTH RELEVANCE: The goal of the proposed work is to understand the molecular mechanisms that cause and support liver specification during development and to explore how the early liver bud cells contribute to the adult organ. Accomplishing these goals will lead to novel insights into liver ontogeny and liver regeneration that will deepen our understanding of what has gone wrong in liver disease, offering new directions for therapeutic design, including the production of hepatocytes from embryonic stem cells.